Thrombus associated diseases such as, for instance, pulmonary embolism (PE), deep-vein thrombosis (DVT), stroke and atherosclerosis are all examples of pathological vascular conditions known to develop in the presence of a clot.
In DVT, as an example, blood clots forming in the deep blood vessels of the legs and groin can block the blood flow from the legs back to the heart. In some occurrences, e.g. embolism, clot particulate is detached and carried by the bloodstream to a blood vessel where it lodges and reduces, or even blocks, the blood flow to vascular tissues. If such a clot lodges in pulmonary blood vessels the embolism can be even fatal.
In the United States alone an estimated 600,000 patients suffer from PE's each year. In approximately 378,000 of these patients, PE goes undetected and approximately 114,000 of these patients later die due to complications associated with the disease. This high mortality is due to the absence of clinical symptoms, in many cases, and to significant limitations associated with currently available methods of investigation and detection.
As the above diseases represent a major cause of mortality, the development of thrombus-specific treatments and detection methodologies is of utmost importance in clinical practice.
In recent years it has been acknowledged that fibrin deposition or accumulation may be associated with various cancer forms, especially solid tumors. The existence of a heterogeneous pattern of fibrin/fibrinogen deposition in various tumor types is a fact supported by a substantial body of correlative and indirect evidence suggesting that fibrin/fibrinogen plays an important role in tumor stroma formation (see, for instance: Costantini V et al., Fibrin and Cancer, Thromb Haemost. 1993; 69:406; Dvorak H F et al. Thrombosis and cancer, Hum Pathol. 1987; 18:275; Dvorak H F et al. Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation, Ann N Y Acad Sci. 1992; 667:101; Cavanagh P G et al., Role of the coagulation system in tumor-cell-induced platelet aggregation and metastasis, Hemostasis. 1988; 18:37; and Bardos H et al., Fibrin deposition in primary and metastatic human brain tumours, Blood Coagul Fibrinolysis. 1996; 7:536).
Several significant hemostatic abnormalities have been also described in patients with cancer including, for instance, disseminated intravascular coagulation, hemorrhagic events and migratory thrombophlebitis. Tumor-mediated activation of the coagulation cascade has been also implicated in both the formation of tumor stroma and the promotion of hematogenous metastasis. Fibrin matrix, moreover, is known to promote the migration of a substantial number of distinct cells types including transformed cells, macrophages and fibroblasts. In particular, much like in a healing wound, the deposition of fibrin/fibrinogen along with other adhesive glycoproteins into the extracellular matrix (ECM) have been shown to serve as a scaffold to support binding of growth factors and to promote the cellular response of adhesion, proliferation and migration during angiogenesis and tumor cells growth (see, for instance: Dvorak H F et al., Vascular permeability factor, fibrin, and the pathogenesis of tumor stroma formation, Ann N Y Acad Sci. 1992; 667:101; Rickles F R et al., Tissue Factor, Thrombin, and Cancer, Chest. 2003; 124:58S-68S; Brown H F et al., Fibrinogen influx and accumulation of cross-linked fibrin in healing wounds and in tumor stroma, Am J Pathol. 1988; 130:4559; Dvorak H F et al., Fibrin containing gels induce angiogenesis: implication for tumor stroma generation and wound healing, Lab Invest. 1987; 57:673; and Rickles F R et al., Tissue Factor, Thrombin and Cancer, Cest. 2003; 124:58S-68S).
Most of the solid tumors, in humans, contain considerable amounts of cross-linked fibrin, thus suggesting its role in tumor stroma formation. To this extent, recent techniques such as immunofluorescence, immunohistochemical and immunoelectron microscopy techniques indicated that fibrin deposition occurs within the stroma of a majority of tumor types and enabled to localize both fibrinogen and fibrin to the tumor-host cell interface (see, for instance: Rickles F R et al, Tissue Factor, Thrombin and Cancer, Cest. 2003; 124: 58S-68S; Costantini V et al., Fibrinogen deposition without trombine generation in primary human breast cancer, Cancer Res. 1991; 51: 349-353; and Simpson-Haidaris P J et al., Tumors and Fibrinogen: The Role of Fibrinogen as an Extracellular Matrix Protein, Ann. N.Y. Acad. Sci., 2001 936(1): 406-425).
Moreover, a correlation seems to exist between plasma fibrinogen levels and tumor size, depth of tumor invasion and metastasis (see, for instance, Lee J H et al., Preoperative plasma fibrinogen levels in gastric cancer patients correlate with extent of tumor, Hepatogastroenterology 2004; 51:1860-3). In addition, it is known that fibrin/platelets are involved in protecting tumor cells from the action of the circulating natural killer units provided by the human immune system, thus improving the survival of circulating tumors (see, for instance, Palumbo J S, et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells, Blood, 2005; 105:178). This implies, for example, that a conventional tumor therapy employing antibodies that target tumors may not be effective in the treatment of fibrin containing tumors as the latter are somehow protected by fibrin itself.
Thus, visualization of fibrin deposition and targeted inhibition/destruction of established vasculature and clotted fibrin is considered an important tool against malignant disease progression. In this respect, there remains the need for improved fibrin-binding compounds to be used in sensitive diagnosis and specific therapy of pathological conditions associated with fibrin deposition, particularly of solid tumors.
The search for thrombus-specific imaging agents began three decades ago when radiolabelled fibrinogen was first evaluated (see, for instance, Kakkar et al., Lancet. 1970, 1:540-542).
A number of MRI based imaging approaches are also described in the literature (see, for instance, Winter P M, et al. Improved molecular imaging contrast agent for detection of human thrombus, Magn Reson Med. August; 2003 50(2):411-6; Botnar R M et al., In vivo molecular imaging of acute and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent, Circulation. 2004, Apr. 27; 109(16):2023-9; Yu X et al., High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent, Magn Reson Med. 2000; 44:867-872; Flacke S et al., Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques, Circulation. 2001; 104:1280-1285).
Radiolabelled platelets and anti-platelet antibodies that bind to forming thrombi, anti-fibrin antibodies, anti-activated platelet antibodies and activated or inactivated tissue type plasminogen activator (tPA) have been also disclosed (see, for instance, Thakur et al., Throm. Res., 1976 9:345-357 and Palabrica et al., Proc. Natl. Acad. Sci., 1989; 86:1036-1040).
Platelet affinity peptides are also known (see, for instance Bautovich et al., J. Nucl. Med., 1994, 35:195-202 and Muto et al., Radiology, 1993; 189 (suppl):303).
The international patent application WO 01/09188 discloses fibrin binding polypeptides useful, when detectably labelled, for localization and imaging of fibrin clots.
WO 02/055544 relates to a class of fibrin-binding polypeptides that are said to be endowed with a lower dissociation rate over those peptides of the aforementioned WO 01/09188, and equally useful for detection, imaging and localization of fibrin-containing clots and, more in general, for the diagnosis/treatment of coronary conditions where fibrin plays a key role.
Among the preferred polypeptides of WO 02/055544 is, for instance, the polypeptide comprising the amino acid sequence WQPCPWESWTFCWDP (see page 9, line 25 of the said international patent application), SEQ ID NO:037, wherein the cysteine “C” residues are believed to form a disulfide bond (see bottom of page 19).
U.S. Pat. No. 5,792,742 discloses fibrin-binding peptides, their preparation and the use of labelled derivatives thereof for the imaging or therapy of clots, thrombi, microthrombi, pulmonary emboli, atherosclerotic lesions or tumors, and for the targeted delivery of therapeutic agents to thrombi, cancer cells and/or sites of given bacterial infections. In vivo test data are therein reported to assess the targeted ability of the claimed peptides towards thrombi.
U.S. Pat. No. 6,991,775 discloses peptide-based multimeric targeted contrast agents that are said to be useful for the diagnosis of thrombosis of deep vein, coronary and carotid, as well as of intracranial, arterial and ventricular aortic thrombi. A wide number of peptide-based MRI contrast agents is disclosed therein and one of them has been used for thrombolysis target assays.
In US 2006/0034773 two AAZA moieties have been conjugated with the fibrin targeted peptides of WO 02/055544 so as to provide an example of biologically active AAZA derivatives. The use of these conjugates for targeting and treating cardiovascular diseases, fibrin containing blood clots/thrombus, plaques or tumors is also claimed.